CN117701738A - SNP molecular marker related to buffalo primordial age traits and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and relates to SNP molecular markers related to the primiparity month age of buffalo reproduction traits. The invention provides 7 SNP (single nucleotide polymorphisms) which are obviously related to the primiparity month-old character (P < 0.01) of the buffalo, and the locus can be applied to genotype analysis of related genes of the primiparity month-old character of the buffalo, provides a new molecular marker resource for auxiliary selection of molecular markers of the primiparity month-old character of the buffalo, and accelerates breeding of fine breed buffalo. The method can directly select individuals on genome level in early stage, does not depend on phenotype information, can obviously improve selection efficiency, quickens breeding process, saves intermediate feeding cost, improves breeding efficiency, reduces breeding cost and has wide application prospect.

Description

SNP molecular marker related to buffalo primordial age traits and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and relates to SNP molecular markers related to the primordial puerperal characters of buffalo and application thereof.

Background

Buffalo is considered by the united nations grain and agriculture organization (FAO) as the livestock with the most development potential and development value, and is an important livestock resource in the south of China. The reproductive traits of buffalo are important economic traits, typically quantitative traits. Due to low genetic force and many influencing factors, breeding of buffalo with high fertility by the traditional method is slow in progress. Currently, whole genome association analysis (GWAS) provides a new direction for the study of quantitative traits, and genetic variation related to economic traits of animals and plants, namely single nucleotide polymorphism (single nucleotidepolymorphism, SNP) can be screened out in the whole genome by GWAS sequencing.

The buffalo is long in generation interval, the primordial age is late (generally, holstein cows produce first embryo before 30 months of age, and cows produce first embryo after about 47 months of age), the early raising cost is high, the return of investment income is long, and the factors restrict the further development of the buffalo milk industry. Therefore, by performing combination analysis of the whole genome sequencing data of buffalo obtained by GBS, the high-quality assembly of buffalo genome (UOA_WB_1) and buffalo reproductive trait (primordial month age), a large number of SNP loci which are not discovered before and are related to the buffalo reproductive trait (primordial month age) can be mined. The understanding of the complex character molecular genetic mechanism is increased, SNP loci influencing the primiparity month age are screened out to carry out gene breeding on buffalo, the primiparity month age of buffalo is reduced, the reproductive capacity of buffalo population is improved, and finally the development of buffalo industry is promoted.

Disclosure of Invention

The invention aims to provide an SNP molecular marker with the characteristics of the primiparity month age of buffalo and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

SNP molecular markers related to buffalo reproductive traits, wherein the reproductive traits refer to the age of buffalo primordia, and the SNP molecular markers related to buffalo primordia at least comprise one of the following markers:

marking: chr4_71082002 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.1, and the polymorphism is C or G;

marking two: chr6-96791388 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.2, and the polymorphism is A or G;

and (3) marking: chr7_59963243 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.3, and the polymorphism is A or C;

and (3) marking: chr10_13862680 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.4, and the polymorphism is C or T;

and fifthly, marking: chr12-3176008 is located at position 51 of the nucleotide sequence shown in SEQ ID NO.5, and the polymorphism is A or G;

and (3) marking: chr13-86238940 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.6, and the polymorphism is A or G;

marking seven: chr18-59367852 is located at position 51 of the nucleotide sequence shown in SEQ ID NO.7, and the polymorphism is A or C.

Another object of the present invention is to provide a primer set for amplifying the above SNP molecular marker combination.

Further described, the primer set comprises a forward primer and a Reverse primer; wherein,

the sequences of the forward primer and the Reverse primer in the primer group marked by the chr4_71082002 molecule are respectively shown as nucleotide sequences SEQ ID NO.8 and SEQ ID NO. 9;

the sequences of the forward primer and the Reverse primer in the primer group marked by the chr6_96791388 molecule are respectively shown as nucleotide sequences SEQ ID NO.10 and SEQ ID NO. 11;

the sequences of the forward primer and the Reverse primer in the primer group marked by the chr7_59963243 molecules are respectively shown as nucleotide sequences SEQ ID NO.12 and SEQ ID NO. 13;

the sequences of Forward primer and Reverse primer in the chr10_13862680 molecular marked primer group are respectively shown as nucleotide sequences SEQ ID NO.14 and SEQ ID NO. 15;

the sequences of the forward primer and the Reverse primer in the primer group marked by the chr12_3176008 molecules are respectively shown as nucleotide sequences SEQ ID NO.16 and SEQ ID NO. 17;

the sequences of Forward primer and Reverse primer in the primer group marked by the chr13_86238940 molecule are respectively shown as nucleotide sequences SEQ ID NO.18 and SEQ ID NO. 19;

the sequences of Forward primer and Reverse primer in the primer group marked by the chr18_59367852 molecule are respectively shown as nucleotide sequences SEQ ID NO.20 and SEQ ID NO. 21.

The third object of the present invention is to provide a kit comprising the primer set as described above.

The fourth object of the invention is to provide the application of the SNP molecular marker combination or the primer set in buffalo molecular marker assisted breeding.

The fourth object of the invention is to provide the application of the SNP molecular marker combination or the primer set in the identification of buffalo reproductive traits.

A fifth object of the present invention is to provide a method for detecting the genotype of buffalo using molecular biology techniques as described above, comprising the steps of: the nucleotide sequences of two flanks of 7 SNP molecular marker loci according to claim 1 are respectively designed into amplification primers, the DNA of a buffalo individual is used as a template for amplification, the amplification products are subjected to first-generation sequencing, and the buffalo individual is subjected to genotyping according to the sequencing result, so that the genotype of the buffalo individual to be detected is identified.

Further described is the application of the method in buffalo molecular marker assisted breeding.

The sixth object of the present invention is to provide a method for breeding a buffalo variety or strain related to the month-old character of buffalo primordium by using the above SNP molecular marker, wherein the method comprises the following steps:

extracting genome DNA of buffalo, detecting 71082002 locus deoxynucleotide of chromosome 4, detecting 71082002 locus deoxynucleotide as C or G, determining that the genotype of buffalo to be detected is CC type, CG or GG type, selecting buffalo with CC type, CG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of buffalo with CC type genes is far lower than that of CG or GG type;

extracting genome DNA of buffalo, detecting 96791388 locus deoxynucleotide of chromosome 6, detecting 96791388 locus deoxynucleotide as A or G, determining that the genotype of the buffalo to be detected is AA type, GA or GG type, selecting the buffalo with AA type, GA or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial month age of the buffalo with GG type and GA type genes is far lower than that of the AA type;

extracting genome DNA of buffalo, detecting 59963243 locus deoxynucleotide of chromosome 7, detecting 59963243 locus deoxynucleotide as A or C, determining that the genotype of buffalo to be detected is AA type, AC or CC type, selecting buffalo with AA type, AC or CC type genes for further seed selection and/or breeding according to breeding requirements, wherein the month age of buffalo initial production of CC type genes is far lower than AA type;

extracting genome DNA of buffalo, detecting 13862680 locus deoxynucleotide of chromosome 10, detecting 13862680 locus deoxynucleotide as T or C, determining that the genotype of the buffalo to be detected is TT type, TC type or CC type, selecting the buffalo with TT type, TC type or CC type genes for further seed selection and/or breeding according to breeding requirements, wherein the month age of buffalo primordial production with TT type or TC type genes is far lower than that of buffalo with CC type;

extracting genome DNA of buffalo, detecting 3176008 locus deoxynucleotide of chromosome 12, detecting 3176008 locus deoxynucleotide as A or G, determining that the genotype of the buffalo to be detected is AA type, AG or GG type, selecting the buffalo with AA type, AG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of the buffalo with AA type or AG type genes is far lower than GG type;

extracting genome DNA of buffalo, detecting 86238940 locus deoxynucleotide of chromosome 13, detecting 86238940 locus deoxynucleotide as A or G, determining that the genotype of buffalo to be detected is AA type, AG or GG type, selecting buffalo with AA type, AG or GG type gene for further seed selection and/or breeding according to breeding requirement, wherein the month age of buffalo with AA type gene is far lower than AG or GG type

Extracting genome DNA of buffalo, detecting 59367852 locus deoxynucleotide of chromosome 18, detecting 593678520 locus deoxynucleotide as A or C, determining that the genotype of buffalo to be detected is AA type, AC or CC type, selecting buffalo with AA type, AC or CC type gene for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of buffalo with AA type or AC type gene is far lower than that of CC type.

By adopting the technical scheme, the invention has the following beneficial effects:

the invention provides 7 SNP (chr4_7108002, chr6_96791388, chr7_59963243, chr10_13862680, chr12_3176008, chr13_86238900, chr18_59367852) of buffalo primordial month age characters, which are obviously related to buffalo primordial month age (P < 0.05), can be applied to genotype analysis of related genes of buffalo primordial month age, and provides new molecular marker resources for auxiliary selection of molecular markers of buffalo high/low primordial month age, and quickens breeding of fine breed buffalo. The method can directly select individuals on genome level in early stage, does not depend on phenotype information, can obviously improve selection efficiency, quickens breeding process, saves intermediate feeding cost, improves breeding efficiency, reduces breeding cost and has wide application prospect.

Drawings

FIG. 1 is a diagram of a primiparity month-old Manhattan of a primiparity month-old trait whole genome association analysis of a buffalo of the present invention. Reference numerals illustrate: relates to the primiparity month-old character of buffalo, which is higher than 7 SNP molecular markers chr4_7108002, chr6_96791388, chr7_59963243, chr10_13862680, chr12_3176008, chr13_86238840 and chr18_59367852 screened by the invention on dotted lines, and the markers are respectively positioned on the chromosomes 4, 6, 7, 10, 12, 13 and 18 of buffalo.

FIG. 2 is a QQ diagram of the genome-wide association analysis of the reproductive trait of buffalo primiparity of the present invention.

Detailed Description

The following is a further description of the specific embodiments of the invention with reference to the accompanying drawings.

Example 1 screening and development of SNP molecular markers for detecting the month-old character of buffalo primordia

(1) The data were from the pedigree, production records and record of reproductive traits of 1 local buffalo, 46 hybrid buffalo, 31 buffalo, 42 mola buffalo (120 total) raised in the chinese guangxi buffalo institute from 2000 to 2021. Characteristic primordial gestation month age is defined as the number of days from birth to first parity of buffalo.

Sample collection and sequencing:

genomic DNA was extracted from blood using phenol/chloroform method. The integrity and yield of genomic DNA was assessed and verified using agarose gel electrophoresis.

Genomic DNA was digested with restriction enzymes, followed by addition of sequencing adaptors with barcode, mixing of samples, construction of small fragment libraries (300-400 bp), and sequencing using the Illumina HiSeqTM 2000 system (Illumina, san Diego, calif.).

(2) Phenotype data arrangement and analysis: the phenotype data collected in step (1) is counted and analyzed by using R software, wherein the phenotype data comprises minimum values, maximum values, average values and standard deviations, and the results are shown in table 1

Table 1 buffalo phenotyping data statistical analysis

	Record number	Mean value of	Standard deviation of	Minimum value	Maximum value	Coefficient of variation
							Age/month of primiparity month	105	44.41	15.50	5.5	120.5	34.9

(3) DNA extraction and sequencing: collecting blood sample from buffalo jugular vein by vacuum hemostix, extracting genome DNA by referring to phenol/chloroform method in molecular cloning experiment guide IV, detecting DNA purity by ultraviolet spectrophotometer, and sending to Guangzhou Diao biotechnology limited company for simplified genome re-sequencing after passing detection, wherein a sequencing platform is Illumina HiSeqTM 2000, filtering sequencing machine data and obtaining effective information, and the filtering standard mainly comprises: 1) Filtering reads containing the linker sequence; 2) When the reads with the N ratio of more than 10% are removed; 3) Removing low-quality reads (the number of bases with the quality value Q less than or equal to 10 accounts for more than 50% of the whole read); after obtaining the effective data, further evaluating the sequencing quality, and checking the error rate distribution condition; sequencing error rates and accuracy are shown in table 2;

TABLE 2 statistical results of buffalo genomic DNA resequencing

Numerical value	Clean Data(bp)	HQ Clean Data(bp)	Q20(％)	Q30(％)	GC(％)
						MIN	676186676	664759766	97.56	92.61	42.11
MAX	2965862268	2892808249	98.58	95.09	43.65
						MEAN	1288934542	1261925441	98.07	93.83	43.01

(4) Genome data alignment: comparing and analyzing genome data obtained by sequencing through biological information analysis software BWA, SAMtools and GATK; the reference genome was a buffalo fourth edition reference gene (GCA_ 003121395.1) (https:// www.ncbi.nlm.nih.gov/datasets/genome/GCF_003121395.1 /), and the filtered reads were aligned to the reference genome using the mem algorithm using alignment software bwa (0.7.12), with the alignment parameter being-k 32-M; results after alignment were marked using picard (1.129) software (markdulicates, et al), but not filtered. The average comparison rate of the population samples is 99.54%. Details are shown in Table 3.

TABLE 3 buffalo genome data alignment statistics

(5) SNP quality control and filtration: variant variation refers to variation at the genomic level caused by insertion or deletion of a single nucleotide or several nucleotides to form a DNA sequence polymorphism. The UnifiedGenotyper module of software GATK (3.4-46) is used for carrying out Variant detection on a plurality of samples of the processed comparison file, the detected variation is filtered by using Variant filtration, and the filtering parameters are-Window 4, -filter 'QD <4.0||FS >60.0||MQ < 40.0', and-G_filter 'GQ < 20'. Through the steps, 2012270 SNPs are obtained preliminarily.

Since rare alleles (very low frequency alleles in the population), high deletion rates, high heterozygosity alleles cause population analysis and whole genome association analysis anomalies (linear models are very weak to handle extreme cases) and can give software erroneous results, the self-written perl script is used for the original marker loci, filtered as follows:

1) Non-allelic sites are removed.

2) Site removal with a second allele frequency (minor allele frequency, MAF) of less than 0.05.

3) Site removal with deletion rate greater than 0.5.

4) Sites with a hybridization ratio greater than 0.8 were removed.

Finally, 691729 SNPs on the autosomes are obtained for subsequent association analysis;

(6) Whole genome association analysis (GWAS): performing principal component analysis by using GCTA software, performing association analysis by using GEMMA software and combining phenotype information and genome SNP information, removing individuals with phenotype values scattered outside the mean value plus or minus 3 times of standard deviation in a certain character, and adding the first three values and sex of the principal component as covariates into a mixed linear model, wherein the model is as follows:

y＝Xα+Zβ+Wμ+e

wherein y is a phenotype vector, X is a genotype matrix, α is a genotype effect vector, Q is a fixed effect matrix (PCA scoring matrix in this study), β is a fixed effect vector, K is a random effect matrix, mainly refers to a genetic relationship matrix, μ is a random effect vector, and e is a residual vector. For each SNP locus, whether alpha is 0 or not is checked, and a probability value p of 0 is used for measuring the association degree of the marker genotype and phenotype, and the smaller the p value is, the smaller the probability of 0 is, and the more likely the marker is associated with the character.

(7) Screening and extracting SNP molecular markers related to the primordial month-old character of buffalo: sites reaching significant association levels were extracted by R software, and the significance threshold was set to Bonferroni-adjusted whole genome suggestive significance (5×10 ^-6 ) To detect meaningful correlations. The information of the sites is shown in figures 1-2 and table 4, and the sites are related to the primiparity month age of buffalo and can be used for breeding the primiparity month age character of buffalo.

When buffalo breeding, the SNP molecular marker can be utilized to design primers on nucleotide sequences of two lateral wings of the buffalo, blood is collected and genome DNA is extracted when the buffalo is born, the primers are utilized to genotype buffalo materials to be tested, individuals with target character genotypes are reserved, and the breeding speed can be increased.

Information of 47 molecular markers

Example 2: application of 7 SNP (Single nucleotide polymorphism) marker locus primer set in detection of buffalo primordial month-old character

The selected buffalo was subjected to jugular vein blood collection and genomic DNA was extracted using a blood genomic DNA extraction kit, and DNA concentration was detected using a nucleic acid concentration meter, and the DNA sample mass was determined by 1% agarose gel electrophoresis. Amplification primers were designed for the 7 candidate SNP sites selected (see Table 5). The primer sequences are as follows:

table 5 7 primer design for candidate SNP loci

Note that: f in the table is an upstream primer Forward primer, R is a downstream primer Reverse primer

PCR amplification was performed using the primers described above, using buffalo blood genomic DNA as a template. A50. Mu.L PCR reaction system was used: ddH2O 19 μL, premix Taq ^TM 25. Mu.L of DNA template 2.0. Mu.L, primers (10. Mu. Mol/L for both the upstream and downstream primers) each 2.0. Mu.L.

PCR reaction conditions:

pre-denaturation at 95 ℃ for 4min; denaturation at 94℃for 10s, annealing for 30s (temperature 55 ℃) and extension at 72℃for 1min for 35 cycles; extending at 72℃for 5min.

The PCR amplified product was purified using a Gel Extraction Kit kit from Shanghai Biotechnology Co., ltd, and specific steps are shown in the kit specification. The PCR purified product obtained above is recovered and directly sent to Huada gene (Shenzhen) Biotech company for first generation sequencing. And (5) genotyping the individual according to the sequencing result. Genotyping results are shown in Table 6.

Table 67 candidate SNP loci at Buffalo genotype frequencies and allele frequencies

It can be seen from Table 6 that the C allele frequency at mutation site chr4-71082002 is significantly greater than the G allele frequency; the G allele frequency of mutation site chr6_96791388 is significantly greater than the A allele frequency; the C allele frequency at the chr7_59963243 locus is significantly greater than the A allele frequency; the T allele frequency at the chr10_13862680 locus is significantly greater than the C allele frequency; the a allele frequencies at chr12_3176008 and chr13_86238940 sites are significantly greater than G; the a allele frequency at chr18_59367852 locus is significantly greater than the C allele frequency.

TABLE 7 verification of significant association SNP for the age of the primiparity of buffalo

The primiparity month age table type value is expressed by 'least square mean value +/-standard deviation', and different letters of the same-column data shoulder marks show significant differences (P < 0.01); the same letters of the shoulder marks or no letter labels indicate that the difference is not significant (P > 0.05); the SNP locus genotypes are sequentially arranged according to mutant type, heterozygous type and reference type. The results show that the candidate chr4_7108002, chr6_96791388, chr7_59963243, chr10_13862680, chr12_3176008, chr13_8623840, chr18_59367852, the candidate chr4_71082002 with a significantly different genotype from buffalo primordial month age (table 7) is found, the chr4_71082002 with a significantly smaller month age than CG or GG, the chr6_96791388 with a significantly smaller month age than AA, the chr7_59963243 with a significantly smaller month age than CC, the chr10_13862680 with a significantly smaller month age than CC, the chr12_3176008 with a significantly smaller month age than GG, the chr13_86238940 with a significantly smaller month age than AG or GG, and the chr18_5936785 with a significantly smaller month age than AA can be used as a marker for breeding to shorten the period of the buffalo primordial, and can be used as a marker for assisting the production of water-borne cows.

Example 4:

during buffalo breeding, the 7 SNP molecular markers can be utilized, amplification primers are respectively designed on nucleotide sequences of two lateral wings of the buffalo, buffalo jugular vein blood is collected at 3-6 months of age, genome DNA is extracted by adopting a blood genome DNA extraction kit, PCR amplification is carried out by taking buffalo individual DNA as a template, first-generation sequencing is carried out on amplified products, genotyping is carried out according to a sequencing result, individuals with target character genotypes are reserved, the breeding age can be shortened, and the breeding speed is increased.

The SNP molecular marker breeding/auxiliary breeding method of buffalo varieties or strains related to the buffalo primordial month-old character can be applied, and the method comprises the following steps:

extracting genome DNA of buffalo, detecting 71082002 locus deoxynucleotide of chromosome 4, detecting 71082002 locus deoxynucleotide as C or G, determining that the genotype of buffalo to be detected is CC type, CG or GG type, selecting buffalo with CC type, CG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of buffalo with CC type genes is far lower than that of CG or GG type;

extracting genome DNA of buffalo, detecting 96791388 locus deoxynucleotide of chromosome 6, detecting 96791388 locus deoxynucleotide as A or G, determining that the genotype of the buffalo to be detected is AA type, GA or GG type, selecting the buffalo with AA type, GA or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial month age of the buffalo with GG type and GA type genes is far lower than that of the AA type;

extracting genome DNA of buffalo, detecting 59963243 locus deoxynucleotide of chromosome 7, detecting 59963243 locus deoxynucleotide as A or C, determining that the genotype of buffalo to be detected is AA type, AC or CC type, selecting buffalo with AA type, AC or CC type genes for further seed selection and/or breeding according to breeding requirements, wherein the month age of buffalo initial production of CC type genes is far lower than AA type;

extracting genome DNA of buffalo, detecting 13862680 locus deoxynucleotide of chromosome 10, detecting 13862680 locus deoxynucleotide as T or C, determining that the genotype of the buffalo to be detected is TT type, TC type or CC type, selecting the buffalo with TT type, TC type or CC type genes for further seed selection and/or breeding according to breeding requirements, wherein the month age of buffalo primordial production with TT type or TC type genes is far lower than that of buffalo with CC type;

extracting genome DNA of buffalo, detecting 3176008 locus deoxynucleotide of chromosome 12, detecting 3176008 locus deoxynucleotide as A or G, determining that the genotype of the buffalo to be detected is AA type, AG or GG type, selecting the buffalo with AA type, AG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of the buffalo with AA type or AG type genes is far lower than GG type;

extracting genome DNA of buffalo, detecting 86238940 locus deoxynucleotide of chromosome 13, detecting 86238940 locus deoxynucleotide as A or G, determining that the genotype of the buffalo to be detected is AA type, AG or GG type, selecting the buffalo with AA type, AG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the month age of buffalo primordial production of the AA type genes is far lower than AG or GG type;

extracting genome DNA of buffalo, detecting 59367852 locus deoxynucleotide of chromosome 18, detecting 593678520 locus deoxynucleotide as A or C, determining that the genotype of buffalo to be detected is AA type, AC or CC type, selecting buffalo with AA type, AC or CC type gene for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of buffalo with AA type or AC type gene is far lower than that of CC type.

The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.

Claims (9)

1. An SNP molecular marker related to buffalo reproduction traits, which is characterized in that: the reproductive trait refers to the age of the buffalo primordia, namely buffalo; the SNP molecular marker related to the age of the primiparity of buffalo at least comprises one of the following markers:

marking: chr4_71082002 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.1, and the polymorphism is C or G;

marking two: chr6-96791388 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.2, and the polymorphism is A or G;

and (3) marking: chr7_59963243 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.3, and the polymorphism is A or C;

and (3) marking: chr10_13862680 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.4, and the polymorphism is C or T;

and fifthly, marking: chr12-3176008 is located at position 51 of the nucleotide sequence shown in SEQ ID NO.5, and the polymorphism is A or G;

and (3) marking: chr13-86238940 is positioned at the 51 st position of the nucleotide sequence shown as SEQ ID NO.6, and the polymorphism is A or G;

marking seven: chr18-59367852 is located at position 51 of the nucleotide sequence shown in SEQ ID NO.7, and the polymorphism is A or C.

2. A primer set for amplifying the SNP molecular marker set according to claim 1.

3. The primer set according to claim 2, wherein: the primer group comprises a forward primer and a Reverse primer; wherein,

the sequences of the forward primer and the Reverse primer in the primer group marked by the chr4_71082002 molecule are respectively shown as nucleotide sequences SEQ ID NO.8 and SEQ ID NO. 9;

the sequences of the forward primer and the Reverse primer in the primer group marked by the chr6_96791388 molecule are respectively shown as nucleotide sequences SEQ ID NO.10 and SEQ ID NO. 11;

the sequences of the forward primer and the Reverse primer in the primer group marked by the chr7_59963243 molecules are respectively shown as nucleotide sequences SEQ ID NO.12 and SEQ ID NO. 13;

the sequences of Forward primer and Reverse primer in the chr10_13862680 molecular marked primer group are respectively shown as nucleotide sequences SEQ ID NO.14 and SEQ ID NO. 15;

the sequences of the forward primer and the Reverse primer in the primer group marked by the chr12_3176008 molecules are respectively shown as nucleotide sequences SEQ ID NO.16 and SEQ ID NO. 17;

the sequences of Forward primer and Reverse primer in the primer group marked by the chr13_86238940 molecule are respectively shown as nucleotide sequences SEQ ID NO.18 and SEQ ID NO. 19;

the sequences of Forward primer and Reverse primer in the primer group marked by the chr18_59367852 molecule are respectively shown as nucleotide sequences SEQ ID NO.20 and SEQ ID NO. 21.

4. A kit comprising the primer set of claim 2 or 3.

5. Use of the SNP molecular marker combination of claim 1 or the primer set of any one of claims 2 or 3 in buffalo molecular marker-assisted breeding.

6. Use of the SNP molecular marker combination of claim 1 or the primer set of any one of claims 2 or 3 in the identification of buffalo reproductive traits.

7. A method for detecting buffalo genotype by utilizing molecular biology technology, which is characterized by comprising the following steps: the nucleotide sequences of two flanks of 7 SNP molecular marker loci according to claim 1 are respectively designed into amplification primers, the DNA of a buffalo individual is used as a template for amplification, the amplification products are subjected to first-generation sequencing, and the buffalo individual is subjected to genotyping according to the sequencing result, so that the genotype of the buffalo individual to be detected is identified.

8. Use of the method of claim 7 in buffalo molecular marker assisted breeding.

9. A method for breeding/assisting in breeding buffalo breeds or lines related to the buffalo primordial month-old traits by using the SNP molecular marker according to claim 1, which is characterized in that the method comprises the following steps:

extracting genome DNA of buffalo, detecting 71082002 locus deoxynucleotide of chromosome 4, detecting 71082002 locus deoxynucleotide as C or G, determining that the genotype of buffalo to be detected is CC type, CG or GG type, selecting buffalo with CC type, CG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of buffalo with CC type genes is far lower than that of CG or GG type;

extracting genome DNA of buffalo, detecting 96791388 locus deoxynucleotide of chromosome 6, detecting 96791388 locus deoxynucleotide as A or G, determining that the genotype of the buffalo to be detected is AA type, GA or GG type, selecting the buffalo with AA type, GA or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial month age of the buffalo with GG type and GA type genes is far lower than that of the AA type;

extracting genome DNA of buffalo, detecting 59963243 locus deoxynucleotide of chromosome 7, detecting 59963243 locus deoxynucleotide as A or C, determining that the genotype of buffalo to be detected is AA type, AC or CC type, selecting buffalo with AA type, AC or CC type genes for further seed selection and/or breeding according to breeding requirements, wherein the month age of buffalo initial production of CC type genes is far lower than AA type;

extracting genome DNA of buffalo, detecting 13862680 locus deoxynucleotide of chromosome 10, detecting 13862680 locus deoxynucleotide as T or C, determining that the genotype of the buffalo to be detected is TT type, TC type or CC type, selecting the buffalo with TT type, TC type or CC type genes for further seed selection and/or breeding according to breeding requirements, wherein the month age of buffalo primordial production with TT type or TC type genes is far lower than that of buffalo with CC type;

extracting genome DNA of buffalo, detecting 3176008 locus deoxynucleotide of chromosome 12, detecting 3176008 locus deoxynucleotide as A or G, determining that the genotype of the buffalo to be detected is AA type, AG or GG type, selecting the buffalo with AA type, AG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of the buffalo with AA type or AG type genes is far lower than GG type;

extracting genome DNA of buffalo, detecting 86238940 locus deoxynucleotide of chromosome 13, detecting 86238940 locus deoxynucleotide as A or G, determining that the genotype of the buffalo to be detected is AA type, AG or GG type, selecting the buffalo with AA type, AG or GG type genes for further seed selection and/or breeding according to breeding requirements, wherein the month age of buffalo primordial production of the AA type genes is far lower than AG or GG type;

extracting genome DNA of buffalo, detecting 59367852 locus deoxynucleotide of chromosome 18, detecting 593678520 locus deoxynucleotide as A or C, determining that the genotype of buffalo to be detected is AA type, AC or CC type, selecting buffalo with AA type, AC or CC type gene for further seed selection and/or breeding according to breeding requirements, wherein the primordial age of buffalo with AA type or AC type gene is far lower than that of CC type.